Assays for detecting the presence and levels of a variety of analytes in body fluid samples are known. Such assays are often designed for simplicity of use so that they can be reliably conducted in a doctor's office or other clinical setting where personnel may have little training in clinical assay procedure or in interpreting assay results. Typically, such assays involve a one-step assay procedure, or employ automated or semi-automated procedures, with the assay reading being determined from a reaction end-point.
One type of diagnostic assay format which is generally adaptable to a one-step assay protocol is an absorptive-pad device, containing a pad or matrix designed to absorb a sample volume, and to produce an analyte-dependent chemical reaction which can be detected on the pad's surface. Examples of absorptive-pad assay devices and methods are described in U.S. Pat. Nos. 3,983,005, 4,069,017, 4,144,306 and 4,447,575.
In general, it is desirable to carry out a body-fluid assay with an undiluted volume. By avoiding sample-dilution, the number of steps in the assay is reduced, and also the possibility of error in interpreting the assay results is minimized. In the case of a blood sample, using an undiluted sample also minimizes the possibility of blood cell lysis and the effect of variations in blood hematocrit on measured analyte concentration.
One limitation of an absorptive-pad assay format, where an undiluted sample is applied, is that the minimum volume of sample needed to wet the pad may contain an amount of analyte that "saturates" the detection range of the assay. In particular, where analyte detection is based on a color change detected at the surface of the reaction pad, the change in color which is detected, e.g., by reflectance absorptiometry, may be produced at a relative low analyte concentration (in contrast to the same reaction in solution, where changes in the concentration of a colored reaction product can be detected over a wide concentration range). Undiluted sample may therefore contain a higher concentration of analyte than can be effectively quantitated in a absorption-pad device.
An example of this limitation has been encountered in absorptive-pad assays for determination of serum cholesterol. This analyte, which is frequently assayed in a clinical laboratory or doctor's office, is clinically important since high blood cholesterol level, and particularly a high level of cholesterol associated with low-density lipoproteins (LDL), is directly associated with a number of serious disease conditions in humans, including coronary artery diseases, biliary obstruction, and liver or thyroid dysfunctions.
Total cholesterol levels in normal individuals is less than 200 mg/dl, although levels as high as 600-700 mg/dl are present in serious hypercholesteremic conditions. In a typical absorptive-pad assay device, employing for example 5-25 .mu.l serum volumes, color saturation of the pad tends may occur above about 300-350 mg/dl, leaving a significant high-cholesterol range which cannot be accurately quantitated.
U.S. Pat. No. 3,907,645 describes a cholesteral assay method in which cholesterol is used to generate H.sub.2 O.sub.2 by reaction with cholesterol oxidase, and the H.sub.2 O.sub.2 is used to generate a visible reaction product by reaction with peroxidase. The patent notes that the amount of colored product produced in the test can be selectively reduced by including increasing amounts of catalase in the assay mixture.
U.S. Pat. No. 4,654,310 proposes the use of a second enzyme or enzyme system to consume analyte or an analyte-produced substrate, to reduce the amount of a reaction product produced by a first enzyme system acting on the analyte. One embodiment of the method employs catalase to compete with peroxidase for consumption of H.sub.2 O.sub.2 generated by analyte and an analyte oxidase.
One limitation of the catalase quenching method proposed in these patents is that the rate of H.sub.2 O.sub.2 decomposition by catalase is typically much faster than H.sub.2 O.sub.2 utilization in product formation. As a result, a catalase concentration which is effective to competitively remove H.sub.2 O.sub.2 at high H.sub.2 O.sub.2 concentration (high analyte concentration) will effectively quench the product formation at relatively low H.sub.2 O.sub.2 concentrations. That is, because the rates of H.sub.2 O.sub.2 -dependent product formation and H.sub.2 O.sub.2 decomposition by catalase are quite different, the amount of quenching produced by catalase will depend on H.sub.2 O.sub.2 concentration. The result is either relatively poor sensitivity at low analyte concentration due to excessive H.sub.2 O.sub.2 decomposition, or relatively little quenching effect at high analyte concentration.
As a further limitation, the relative amount of H.sub.2 O.sub.2 quenching in the assay system would depend on the relative stabilities of catalase and the analyte-specific enzyme(s) used in the assay.